Resolution and repair of acute lung injury by macrophage-derived iNOS

巨噬细胞衍生的 iNOS 解决和修复急性肺损伤

• 批准号: 8539070
• 负责人: Franco R D'Alessio
• 金额: $ 23.14万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-09 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8539070
• 关键词: Acute; Acute Lung Injury; Adenoviruses; Adoptive Transfer; Adult Respiratory Distress Syndrome; Albumins; Alveolar; Alveolar Cell; Alveolar Macrophages; Animals; Antibodies; Attenuated; Blood capillaries; Bone Marrow; Cell Communication; Cells; Chimera organism; Clinical; Coupled; Edema; Epithelial; Event; Extravasation; Flow Cytometry; Gene Delivery; Generations; Gram-Negative Bacteria; Healthcare Systems; ITGAM gene; Immune response; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Injury; Life; Lipopolysaccharides; Lung; Lung Inflammation; Lymphocyte; Mediating; Mediator of activation protein; Modeling; Molecular; Mus; Nitrates; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitrites; Outcome; Pathogenesis; Patients; Pattern; Permeability; Phenotype; Play; Process; Production; Protein Isoforms; Proteins; Recovery; Regulation; Regulatory T-Lymphocyte; Relative (related person); Reporting; Resolution; Role; Signaling Molecule; Staging; Stimulus; TNF gene; TNFRSF5 gene; Time; Wild Type Mouse; abstracting; capillary; cytokine; human NOS2A protein; improved; in vivo; injured; killings; lung injury; lung repair; macrophage; monocyte; mortality; mouse model; neutrophil; novel; protein expression; repaired; response

Project Summary/Abstract While early events in the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) have been defined, little is known about mechanisms mediating resolution. To begin to search for potential determinants of resolution, we exposed wild type (WT) mice to intratracheal lipopolysaccharide (i.t. LPS) and assessed the response at intervals out to day 10, a time when injury had resolved. Bronchoalveolar (BAL) and lung mediators increased after i.t. LPS, among them BAL nitrites/nitrates (NOx). Consistent with the increase in NOx, we found that inducible nitric oxide synthase (iNOS) protein expression was significantly upregulated in the lung and peaked at day 4 after injury. Early lung injury was attenuated in iNOS-/- mice after i.t. LPS, however recovery by day 10 was markedly impaired in comparison to WT mice. iNOS-/- mice had increased mortality as well as persistently elevated BAL protein, albumin, cells, neutrophils and pro- inflammatory cytokines. Adoptive transfer of WT iNOS+/+ bone marrow-derived monocytes or direct adenoviral gene delivery of iNOS into injured iNOS-/- mice (given 1 and 2 days after i.t. LPS respectively) restored resolution of ALI in a pattern similar to WT; in contrast, transfer of iNOS-/- monocytes or delivery of sham adenovirus did not achieve resolution. To begin to understand how iNOS contributed to resolution of ALI, we performed multicolor flow cytometry of alveolar cells at intervals after i.t. LPS. We observed markedly decreased numbers of Regulatory T cells (Tregs) and a sustained expression of macrophage/monocyte co- signalling molecules (e.g. CD86 and CD40) in iNOS-/- mice compared to WT mice out to day 7 after injury. Transfer of supplemental WT Tregs or antibody-mediated blockade of CD86 in injured iNOS-/- mice remarkably restored resolution of lung injury. We hypothesize that macrophage-derived iNOS plays a pivotal role in mediating resolution of ALI by modulating immune responses in the lung, thus facilitating clearance of alveolar inflammation and promoting lung repair. To move these findings towards consideration in a clinical context, we propose the following specific aims: 1. To identify mechanisms by which iNOS modulates macrophage innate immune responses in the lung after injury. Using in vivo and in vitro approaches, we will determine the role of iNOS in modifying macrophage/monocyte innate immune responses at different stages of the injury resolution response. iNOS- mediated effects on macrophage co-signalling molecules and its relative contribution in resolution of ALI will be examined by using antibody blockade and creation of bone-marrow chimeras. We will compare the effects of exogenous vs. endogenous NO in modulating macrophage immune responses after injury. 2. To determine the role of iNOS in modulating alveolar macrophage-Regulatory T cell interactions to promote resolution of ALI. In vivo and in vitro studies will be used to evaluate the role of macrophage- derived iNOS in modulating Treg lymphocyte phenotype and function. The potential effects of Treg-derived iNOS will be also be investigated. 3. To determine if manipulation of iNOS can improve outcomes in different models of ALI. We will use direct adenoviral gene delivery of iNOS or NO donors in vivo, both to restore resolution of ALI in iNOS-/- mice after i.t. LPS and direct live gram negative bacteria model of ALI. Targets identified in SA 1 and 2 (e.g. Co- signalling molecules) will be manipulated in an attempt to accelerate resolution in 'severely' injured WT animals. Definition of mechanisms responsible for resolution of ALI may provide novel targets for therapy in an important clinical condition which kills more than 75,000 people annually, and for which therapy at present remains supportive. We believe targeting iNOS expression, particularly at later stages after onset of ALI, may prove useful as therapy to accelerate resolution in patients with ALI.

项目摘要/摘要 而急性肺损伤(ALI)和急性呼吸窘迫综合征的发病机制中的早期事件 (ARD)已经定义，但对调解解决的机制知之甚少。开始寻找 为了确定可能的决定因素，我们将野生型(WT)小鼠暴露于气管内脂多糖(I.T. LPS)，并每隔10天评估一次反应，这一时间段伤情已痊愈。支气管肺泡 (BAL)和肺组织介质在I.T后升高。LP，其中BAL亚硝酸盐/硝酸盐(NOx)。与 NOx增加时，我们发现诱导型一氧化氮合酶(INOS)蛋白表达明显增加 肺组织中表达上调，伤后4d达高峰。INOS-/-小鼠早期肺损伤减轻 IT。然而，与WT小鼠相比，第10天的恢复明显受损。INOS-/-小鼠有 死亡率增加以及BAL蛋白、白蛋白、细胞、中性粒细胞和促红细胞生成素持续升高。 炎性细胞因子。WT iNOS+/+骨髓来源单核细胞过继转移或直接腺病毒转移 诱导型一氧化氮合酶基因导入受损的诱导型一氧化氮合酶-/-小鼠(注射后1天和2天)LP分别)已恢复 ALI的溶解模式类似于WT；相反，iNOS-/-单核细胞的转移或假手术 腺病毒没有达到溶解的目的。为了开始了解iNOS如何有助于ALI的解决，我们 每隔一段时间行肺泡细胞多色流式细胞术。LP。我们明显地观察到 调节性T细胞(Treg)数量减少和巨噬细胞/单核细胞协同表达 与WT小鼠相比，iNOS-/-小鼠的信号分子(如CD86和CD40)在损伤后第7天出现。 补充WT Tregs或抗体介导的CD86阻断在损伤的iNOS-/-小鼠中的显著转移 肺损伤恢复正常。我们假设巨噬细胞来源的诱导型一氧化氮合酶在 通过调节肺内免疫反应调节ALI的消退，从而促进肺泡的清除 发炎，促进肺修复。为了推动这些发现在临床上得到考虑，我们 提出以下具体目标： 1.确定iNOS调节小鼠巨噬细胞先天免疫反应的机制 伤后肺损伤。使用体内和体外方法，我们将确定iNOS在修饰中的作用 巨噬细胞/单核细胞先天免疫反应在损伤分解反应的不同阶段。伊诺斯- 巨噬细胞共信号分子的介导作用及其在ALI消退中的相对贡献 通过抗体阻断和建立骨髓嵌合体进行检测。我们将比较以下几种方法的效果 外源性与内源性NO对创伤后巨噬细胞免疫反应的调节作用。 2.确定iNOS在调节肺泡巨噬细胞-调节性T细胞相互作用中的作用 推动ALI的化解。体内和体外研究将被用来评估巨噬细胞- 诱导型一氧化氮合酶对Treg淋巴细胞表型和功能的调节作用Treg衍生的潜在效应 INOS也将接受调查。 3.确定iNOS能否改善不同ALI模型的预后。我们将使用 体内直接携带iNOS或NO供体的腺病毒基因，均可恢复iNOS-/-小鼠对ALI的溶解 在IT之后。脂多糖和直接活革兰氏阴性菌ALI模型。SA 1和SA 2中确定的目标(例如 信号分子)将被操纵，以试图加速WT的严重损伤的解决 动物。 明确ALI的解决机制可能为急性肺损伤的治疗提供新的靶点 每年导致75,000多人死亡的重要临床疾病，目前的治疗方法 仍然表示支持。我们认为靶向iNOS的表达，特别是在ALI发病的后期，可能 事实证明，这是一种有效的治疗方法，可以加速ALI患者的病情缓解。